Methods of screening for compounds that modulate TAFIa activity, compounds, and methods of using the compounds

ABSTRACT

Provided are methods of screening compounds for any aspirin-related activity other than TAFI inhibition, and also for non-inhibition of TAFI. Compounds identified by the screening methods can be used to treat, prevent or manage in a patient pain, fever, colon cancer, pancreatic cancer or an inflammatory, platelet aggregation, fibrinolytic or hemorrhagic disease or disorder. Also provided is a method of evaluating test compounds for TAFI inhibitory activity wherein the TAFI inhibitory activity of these test compounds is compared to the TAFI inhibitory activity of aspirin or its derivatives or metabolites. Further provided is a method of treating, preventing or managing in a patient, a hemorrhagic or thrombotic disease or disorder with high dose aspirin or aspirin derivatives or metabolites. Also contemplated is a method of treating, preventing or managing in a patient, pain, fever, colon cancer, pancreatic cancer or an inflammatory, platelet aggregation, fibrinolytic or hemorrhagic disease or disorder comprising administering aspirin or a derivative thereof or any other therapeutic having at least one desired therapeutic or prophylactic activity of aspirin to a patient in need thereof and administering to the patient a factor that promotes TAFIa activity, e.g. stabilized TAFIa, to ameliorate one or more adverse side effects of the therapeutic. Compounds identified by the methods of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.60/407,138, filed on Aug. 29, 2002 and to U.S. application Ser. No.60/407,395, filed on Aug. 30, 2002.

The foregoing applications, as well as all documents cited in theforegoing applications (“application documents”) and all documents citedor referenced in the application documents are hereby incorporated byreference. Also, all documents cited in, this application (“herein-citeddocuments”) and all documents cited or referenced in the herein-citeddocuments are incorporated by reference. In addition, any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the application documents or herein-cited documents are incorporatedby reference. Documents incorporated by reference into this text or anyteachings therein can be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

FIELD OF THE INVENTION

The invention relates to the general field of fibrinolysis. One aspectof the invention relates to methods of screening compounds for anyaspirin-related activity other than TAFI inhibition, e.g., analgesia,fever reduction, anti-inflammation, anti-platelet aggregation, coloncancer prophylaxis, pancreatic cancer prophylaxis, but fornon-inhibition of TAFI. A second aspect of the invention encompassesutilizing the compounds identified by the screening methods of theinvention to treat, prevent or manage in a patient pain, fever, coloncancer, pancreatic cancer or an inflammatory, platelet aggregation,fibrinolytic or hemorrhagic disease or disorder. A third aspect of theinvention relates to a method of evaluating test compounds for TAFIinhibitory activity wherein the TAFI inhibitory activity of these testcompounds is compared to the TAFI inhibitory activity of aspirin or itsderivatives or metabolites. A fourth aspect of the invention comprises amethod of treating, preventing or managing in a patient, a hemorrhagicor thrombotic disease or disorder with high dose aspirin or aspirinderivatives or metabolites, including but not limited to salicylic acid,salicylic acid, gentisic acid, glycyl amides and α-hydroxybenzoatederivatives. A fifth aspect of the invention relates to a method oftreating, preventing or managing in a patient, pain, fever, coloncancer, pancreatic cancer or an inflammatory, platelet aggregation,fibrinolytic or hemorrhagic disease or disorder comprising administeringaspirin or a derivative thereof or any other therapeutic having at leastone desired therapeutic or prophylactic activity of aspirin to a patientin need thereof and administering to the patient a factor that promotesTAFIa activity, e.g. stabilized TAFIa, to ameliorate one or more adverseside effects of the therapeutic. A sixth aspect of the invention relatesto compounds identified by the methods of the invention.

BACKGROUND OF THE INVENTION Aspirin and its Activities

A proper balance between the activities of coagulation and fibrinolyticcascades is needed both to protect an organism from excessive blood lossupon injury and to maintain blood flow within the vascular system. Thetwo opposing coagulation and fibrinolytic cascades are recognized tocomprise a series of zymogen to enzyme conversions which terminate inthe two respective proteolytic enzymes, thrombin and plasmin. Theseenzymes catalyze the formation and removal of fibrin within thecirculatory system. Imbalances are characterized by either bleeding orthrombotic tendencies which may result in heart attacks or strokes inthe organism.

The anti-thrombotic effect of aspirin has long been recognized, and lowdoses of aspirin (acetylsalicylic acid; ASA) are recommended for theprevention of ischemic events in patients with coronary artery disease.In large clinical studies, aspirin has been shown to significantlyreduce both the occurrence of myocardial infarction and mortality ratein patients with unstable angina pectoris that often occurs before aheart attack and/or previous myocardial infarction. Simoons, M., Lancet,2001, 357(9272):1915-24.

The action of aspirin as well as other non-steroidal anti-inflammatorydrugs (NSAIDS) is thought to derive mostly from the selective inhibitionof cyclooxygenase 1 and 2 (COX-1 and -2). At the platelet-vessel wall,aspirin at low doses selectively and irreversibly inhibits COX-1, whichsynthesizes thromboxane A2 (Tx-A2) and causes platelet activation.Hence, aspirin prevents blood platelets from aggregating, one of theinitial steps in the formation of blood clots.

Aspirin is also a currently available therapy for several other diseasesand disorders including fever, colon cancer, pancreatic cancer andinflammatory diseases such as arthritis. Aspirin is often a first-linetherapy for rheumatoid arthritis, an inflammatory process which causeserosion or destruction of bone and cartilage joints. In addition toaspirin, current arthritis treatment regimens often employ ibuprofen andCOX-2 inhibitors such as CELEBREX® (celecoxib) and VIOXX® (rofecoxib)which also act by reducing inflammation of the joints. Although thesetreatments are well established for arthritis sufferers, they often haveunwanted or adverse side effects.

Thus, while aspirin and other drugs are often used for their knownanalgesic activity, antipyretic activity, anti-inflammatory activity,anti-platelet aggregation activity and prophylactic properties,aspirin's mechanism of action, and concomitantly, the mechanisms thatlead to the production of unwanted side effects, are not yet fullyunderstood. For example, the anti-platelet activity of aspirin cannotaccount for all the biological effects associated with aspirin therapy.Aspirin has been shown to reduce thrombin generation at the site ofinjury and can effect tissue factor-initiated coagulation. Undas et al.,2001, Blood, 98:2423-2431. Other studies suggest that aspirin therapyincreases the fibrinolytic activity of plasma. Green, D., 2001, Clin.Cornerstone, 3:50-60. Further studies have been unable to attribute themechanism by which aspirin enhances fibrinolysis to changes in tissueplasminogen activator levels. Bjomsson et al., 1989, J. Pharmacol. Exp.Ther., 250:154-161. Thus, there is confusion in the art regarding themechanism of aspirin action, particularly in causing adverse sideeffects.

Thrombin Activatable Fibrinolysis Inhibitor

Thrombin activatable fibrinolysis inhibitor (“TAFI”) is a 60 kDaglycoprotein present in human plasma that modulates fibrinolysis invivo. TAFI present in plasma is a proenzyme form which is mostefficiently activated by proteolytic cleavage at Arg-92 with athrombin-thrombomodulin complex. TAFI may also be activated byproteolytic cleavage by other proteolytic enzymes including, but notlimited to, thrombin or plasmin (“activation of TAFI”). Upon activationof TAFI by proteolytic cleavage with thrombin-thrombomodulin, an activeenzyme of 35 kDa is formed with carboxypeptidase-like activity(“TAFIa”). This molecule has also been referred to in the literature asplasma carboxypeptidase B (“PCPB”), or plasma carboxypeptidase U(“PCPU”). Tan et al., Biochemistry, 1995, 34:5811-5816; Wang et al., J.Biol. Chem. 269:15937 (1994); Nesheim et al., 1995, J. Biol. Chem. 270:14477.

Modulation of fibrinolysis occurs when TAFIa cleaves C-terminal arginineand lysine residues of partially degraded fibrin, thereby inhibiting thestimulation of tissue plasminogen activator (t-PA) modulated plasminogenactivation. The fibrinolytic system is activated primarily by t-PA whichis provided by damaged cells in the blood vessel wall. t-PA convertscirculating plasminogen to the active protease plasmin and can produceeither slow enhancement of fibrinolysis or, when combined with fibrin,rapid enhancement of fibrinolysis. The effect of t-PA on fibrinolysiscan be blocked by a class of inhibitors termed plasminogen activatorinhibitors (PAIs), of which several have been identified.

Thrombomodulin is a component of the blood vessel wall which bindsthrombin and changes its specificity from fibrinogen to protein C,resulting in a molecule possessing anticoagulant, rather thanprocoagulant, activity. The thrombin-thrombomodulin complex catalyzescleavage of protein C to activated protein C, which results indown-regulation of the coagulation cascade by proteolyticallyinactivating the essential cofactors, Factor Va and VIIIa. In thismanner, the body regulates coagulation cascade.

Studies such as that by Taylor et al., 1985, Thromb. Res. 37:639 havesuggested that activated protein C is not only an anticoagulant, butalso profibrinolytic, both in vivo and in vitro. Subsequently, it wasdetermined that protein C only appears profibrinolytic because itprevents the thrombin-catalyzed activation of a previously unknownfibrinolysis inhibitor, whose precursor was isolated from plasma anddesignated as being TAFI.

TAFI was discovered independently in three different laboratories. Itinitially appeared as an unstable carboxypeptidase B-like molecule inhuman serum and was described by Hendriks et al., 1990, Biochim.Biophys. Acta 1014:86. A year later the cDNA for the molecule wascloned, its amino acid sequence was described, its activation by trypsinand its enzymatic properties toward synthetic carboxypeptidase Bsubstrates was reported (see U.S. Pat. No. 5,206,161). In 1994, Wang etal., (1994, J. Biol. Chem. 269:15937) isolated the activated moleculeand named it carboxypeptidase U (“U” being designated for unstable).Subsequently, Nesheim et al. (1995, J. Biol. Chem. 270:14477) showedthat the protein was both activated by thrombin and inhibitsfibrinolysis, and designated the molecule TAFI. The co-identity of PCPB,PCPU, and TAFI has been established by their independent chromatographicbehavior on plasminogen Sepharose® and the amino acid sequences presentat the activation cleavage site.

The mechanism of TAFI inhibition of fibrinolysis can be schematicallydescribed as depicted in FIG. 5.

As TAFIa is believed to play a central regulatory role in thefibrinolytic cascade, the manipulation of TAFIa levels or activity inbiological fluids has important therapeutic applications with respect tohemorrhagic disorders including, but not limited to, vascular and heartpathologies, and stroke. Inhibitors of TAFIa enhance fibrinolysis andhave an anti-coagulant effect (see U.S. Pat. No. 5,993,815). Inhibitorsof TAFIa could also be effective at treating or preventing theinflammation associated with arthritis as vascular endothelial growthfactor (VEGF) is a potential substrate of TAFIa. VEGF has been linkedwith arthritis (Farva, R. A., 1994, J. Exp. Med. 180:341-6).

There remains a need in the art for methods of modulating TAFIa activityfor therapeutic use in the treatment, prevention or management ofhemorrhagic or thrombotic diseases or disorders. Moreover, there is aneed to identify compounds that have the analgesic, antipyreticactivity, anti-inflammatory activity, anti-platelet aggregation activityand prophylactic activity of aspirin without the hemorrhagic sideeffects. In addition there remains a need in the art for effectivemethods of screening compounds for TAFIa modulating activity which maybe used in the treatment of fibrinolytic or thrombotic diseases ordisorders.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the surprising discoverythat aspirin and two of its derivatives, salicylic acid (“SA”) andsalicyluric acid (“SU”), promote fibrinolysis by inhibiting TAFIaenzymatic activity and, in the case of salicylic acid and salicyluricacid, by inhibiting activation of TAFI, i.e., the conversion of TAFIproenzyme to TAFIa. The present inventors have identified the mechanismmediating aspirin's hemorrhagic side effects, and thus a novel targetfor aspirin action. In brief, the present invention relates to methodsfor diagnostic and/or therapeutic-use of TAFIa inhibitors, and methodsof screening and modeling compounds for TAFIa inhibitory activity, orlack thereof, and other aspirin activities, or lack thereof. Theinvention further relates to compounds identified by the methods ofscreening discovered by the inventors.

The compounds screened and identified by the methods of the inventioncan be of the general formula of Structure I:

where

-   R1 is H, OH, alkoxy, aralkyloxy, NH₂, acylamino or CH₂N(alkyl)₂;-   R2 is H, OH, alkoxy, aralkyloxy, NH₂, acylamino or CH₂N(alkyl)₂;-   R3 is H, aryl or aralkyl;-   R4 is H or R4+R5 is O or NH;-   R5 is H or R4+R5 is O or NH;-   R6 is H or COOH;-   x is CH₂, NH or O;-   Y is CH₂, NH or O;-   Z is (CH₂)_(n)NH₂, NHCOAr, NHC(═NH)NH₂, (CH₂)_(p)NHC(═NH)NHSO₂Aryl,    C(═NH)NH₂ or S₂(CH₂)₂NH₂;-   n is 1, 2, 3, 4, 5 or 6;-   m is 1, 2, 3, 4, 5 or 6; and-   p is 0, 1, 2 or 3.

The compounds screened and identified by the methods of the inventioncan also be of the general formula of Structure II:

where:

-   R1 is H, OH, alkoxy, aralkyloxy, NH₂, acylamino or CH₂N(alkyl)₂;-   R2 is H, OH, alkoxy, aralkyloxy, NH₂, acylamino or CH₂N(alkyl)₂;-   R3 is alkyl, aryl or aralkyl;-   R4 is OH or COOH; and-   n is 0, 1, 2, 3, 4 or 5.

The compounds screened and identified by the methods of the inventioncan also be of the general formula of Structure III:

where:

-   R1, R, R5, R6 is H, OH, alkoxy, aralkyloxy, NH₂, acylamino or    CH₂N(alkyl)₂;-   R3 is H or R3+R4=O or NH;-   R4 is H or R3+R4=O or NH;-   X is O, NH or CH₂;-   Y is O, NH or CH₂;-   n is 0, 1, 2, 3 or 4; and-   m is 0, 1, 2, 3 or 4.

In one embodiment of the invention, compounds are screened for bothaspirin activities and non-inhibition of TAFIa, to identify compoundswith the more beneficial activities of aspirin, but without certainunwanted or adverse side effects, e.g. bleeding. These compounds can beused at higher doses and/or longer time periods than aspirin and itsderivatives, since these unwanted or adverse side effects of aspirinwould be eliminated. In one embodiment, a test compound is screened forCOX-1 and/or COX-2 activity and for non-inhibition of TAFIa. In anotherembodiment, test compounds are screened for at least one aspirinactivity selected from the group consisting of analgesia, feverreduction, anti-inflammation, anti-platelet aggregation, colon cancerprophylaxis, pancreatic cancer prophylaxis and thrombotic diseaseprophylaxis, and for non-inhibition of TAFIa activity and/ornon-inhibition of TAFI activation. In another embodiment, test compoundsare screened for more than one aspirin activity, other than inhibitionof TAFI activation or TAFIa activity, selected from the group consistingof analgesia, anti-inflammation, anti-platelet aggregation, feverreduction, colon cancer prophylaxis, pancreatic cancer prophylaxis andthrombotic disease prophylaxis, and for non-inhibition of TAFIa activityand/or non-inhibition of TAFI activation.

Another aspect of the invention is directed to methods of screeningcompounds for TAFIa inhibitory activity and/or inhibition of TAFIactivation. In a particular embodiment, TAFIa inhibitory activity of thetest compound is compared to that of aspirin, salicylic acid,salicyluric acid or gentisic acid. Test compounds can be screened forTAFIa inhibitory activity and for non-inhibition of aspirin activity.

A further aspect of the invention involves compounds identified by themethods of screening. In particular, several compounds have beenidentified that have TAFIa inhibitory activity. One of these is thecompound designated L278R, having the following structure:

Another compound of the invention is designated. LT32, and has thefollowing structure:

A further compound of the invention is designated JV59, and has thefollowing structure:

Other aspects of the invention relate to methods of treating, preventingor managing pain, fever, inflammation, platelet aggregation, coloncancer or pancreatic cancer comprising administering a therapeuticallyor prophylactically effective amount of a compound identified by thescreening methods of the invention to a patient in need thereof. Aparticular embodiment of the invention relates to a method of treating,preventing or managing hemorrhagic or thrombotic diseases or disorderscomprising administering a therapeutically or prophylactically effectiveamount of a TAFIa modulator, or a pharmaceutically acceptablecomposition thereof, to a patient having such a disease or disorder. Inpreferred embodiments of the invention, aspirin-derived inhibitors ofTAFIa include, but are not limited to salicylic acid, salicyluric acid,gentisic acid, glycyl amides and α-hydroxybenzoate derivatives.

A further aspect of the invention relates to limiting the adverseeffects of aspirin therapy by administering a factor that promotes TAFIaactivity, such as stabilized TAFIa. See U.S. patent application Ser. No.10/116,095, filed Apr. 4, 2002. One embodiment of the invention relatesto methods of treating, preventing or managing pain, fever,inflammation, platelet aggregation, colon cancer or pancreatic cancercomprising the steps of administering a therapeutically orprophylactically effective amount of aspirin or aspirin derivative orother anti-inflammatory to a patient in need thereof and administeringto a patient an effective amount of stabilized TAFIa.

The compounds of the invention can be formulated into pharmaceuticalcompositions and administered in combination with another prophylacticor therapeutic agent. In certain embodiments, the other therapeutic orprophylactic agent is useful for the treatment, prevention or managementof at least one of the following: pain, fever, inflammation, plateletaggregation, fibrinolysis or hemorrhage, colon cancer or pancreaticcancer. When used in combination with other prophylactic and/ortherapeutic agents, the compound of the invention, or pharmaceuticalcomposition thereof, can be administered prior to, subsequent to orconcurrently with the other therapeutic or prophylactic agents.Additionally, agents used in combination may be administered in the samecomposition or in separate pharmaceutical compositions.

These and other aspects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference, in which:

FIG. 1 shows the effect on TAFIa activity of incubation with variousconcentrations of salicylic acid (“SA”) (open circles), salicyluric acid(2-hydroxyhippuric acid) (“SU”) (closed squares) and acetylsalicylicacid (ASA) (closed circles) for 1 hour at room temperature. ResidualTAFIa activity was measured using ActiFLUOR TAFIa. FIG. 1 shows thatTAFIa carboxypeptidase activity is inhibited in a dose-dependent mannerby ASA, SA and SU. SU was the most effective inhibitor followed by SAand ASA.

FIG. 2 shows the effect on TAFIa activity of incubation with differentconcentrations 0 μg (open circles), 200 μg (closed circles), 400 μg(open squares) and 800 μg (closed squares) of salicylic acid at 37° C.,wherein aliquots were later removed and measured for TAFIa activity.

FIG. 3 shows the effect on TAFIa activity of incubation with variousconcentrations of salicylic acid at room temperature for 1 hour.Residual TAFIa activity was measured using ActiFLUOR TAFIa. FIG. 3 showsthat TAFIa activity is inhibited in a dose-dependent manner by SA uponincubation at room temperature. About 60% TAFIa activity is lost at 25mM SA.

FIG. 4 shows TAFI activation by thrombin/thrombomodulin in the presenceof various compounds. After 20 minutes, aliquots were removed andanalyzed by SDS-PAGE. Lane 1. Acetylsalicylic acid; 2. Salicylic acid;3. 4-hydroxybenzoic acid; 4. 2-hydroxybenzoic acid (salicyluric acid);5. Ibuprofen; 6. Acetominophen; 7 Control with no addition; 8. TAFI. Theresults in FIG. 4 indicate that the salicylic acid and 2-hydroxybenzoicacid (salicyluric acid) inhibit the activation of TAFI. Interestingly,ASA and the other salicylate compounds tested did not appear to have asignificantly effect on the rate of activation of TAFI bythrombin/thrombomodulin. Without being bound by any theory, theseresults, suggest that SA ad SU directly blocks the activation site ofTAFI or indirectly blocks TAFI activation possibly throughconformational effects upon binding to TAFI.

FIG. 5 is a schematic illustration of the mechanism of TAFI inhibitionof fibrinolysis.

FIG. 6 shows competitive inhibition of TAFIa by gentisic acid. Differentconcentrations of gentisic acid (GA) were incubated with TAFIa andActichrome 874 TAFI Developer. TAFI activity (Vmax) was determined.

FIG. 7 shows increased-stability at room temperature of stabilized TAFIacompared to conventional TAFIa.

FIG. 8 shows concentration-dependent inhibition of TAFIa activity byL278 using two methods. Method 1: no preincubation (□); Method 2.Preincubation for 1 hour at 4° C. prior to assay (▴). Curve fitting wasperformed using Softmax Pro program.

FIG. 9 shows concentration-dependent inhibition of TAFIa activity byLT32. The results from three independent experiments are shown. Curvefitting was performed using Softmax program.

FIG. 10 shows concentration-dependent inhibition of TAFIa activity byJV59. The results from two independent experiments are shown. Curvefitting was performed using Softmax program.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although a number of methodsand materials similar or equivalent to those described herein can beused in the practice of the present invention, the preferred materialsand methods are described herein.

The term “derivative” as used herein refers to a chemical compoundrelated structurally to another chemical compound and therapeuticallyderivable from it. “Aspirin derivatives” or “derivatives of aspirin”include, but are not limited to salicylate, salicylatic acid, glucuronicacid, dihydroxyamids and salsalate (a dimer of salicylic acid).Additionally, derivatives of α-hydroxybenzoate are also contemplated bythe instant invention, including but not limited to gentisic acid andglycyl amides.

The term “metabolite” as used herein refers to a chemical compoundproduced by chemical conversion of an administered chemical compound ina living organism.

The term “small molecule” as used herein refers to a molecule with amolecular weight less than 500.

The phrase “modulation of fibrinolysis” as used herein means theinhibition or activation of the fibrinolytic cascade which may have ananti-coagulant or procoagulant effect.

“TAFI inhibitor” or “inhibitor of TAFI” as used herein means anymolecule that blocks, reduces or retards the activation of TAFI, i.e.,any molecule that blocks, reduces or retards the cleavage of fibrin orother substrates by TAFIa, any molecule that blocks, reduces, or retardsTAFIa enzymatic activity or any molecule that blocks, reduces or retardsthe cleavage of TAFI proenzyme to form TAFIa.

The phrase “stabilized TAFIa” as used herein means TAFIa that has ahalf-life of at least one hour at room temperature, more preferably ofat least two hours, more preferably of at least four hours, morepreferably of at least eight hours, more preferably of at least twelvehours, more preferably of at least twenty-four hours and most preferablyof at least forty-eight hours. Prepared, e.g., using methods of U.S.patent application Ser. No. 10/116,095, filed Apr. 4, 2002.

The terms “disorder” and “disease” are used interchangeably to refer toa condition in a patient. In particular, the term “thrombotic disease”is used interchangeably with the term “thrombotic disorder” to refer toa condition in a patient characterized by undesired clot formation. Inparticular, the term “fibrinolytic disease” is used interchangeably withthe term “fibrinolytic disorder” to refer to a condition in a patientcharacterized by enhanced or reduced dissolution of fibrin clot. Inparticular, the term “inflammatory disease” is used interchangeably withthe term “inflammatory disorder” to refer to a condition in a patientcharacterized by inflammation, preferably chronic inflammation. One ofskill in the art is capable of recognizing and diagnosing thrombotic,fibrinolytic and inflammatory diseases and disorders.

The term “pain” refers to all categories of pain, including pain that isdescribed in terms of stimulus or nerve response, e.g., somatic pain(normal nerve response to noxious stimulus) and neuropathic pain(abnormal response of an injured or altered sensory pathway, oftenwithout clear noxious input); pain that is categorized temporally, e.g.,chronic pain and acute pain; pain that is categorized in terms ofseverity, e.g., mild, moderate, or severe; pain that described in termsof origin or site of inflammation, e.g., peripheral nervous system orcentral nervous system; and pain that is a symptom or a result of adisease state or syndrome, e.g., inflammatory pain, cancer pain, AIDSpain, arthropathy, migraine, trigeminal neuralgia, cardiac ischaemia,and diabetic neuropathy (see e.g., Harrison's Principles of InternalMedicine, pp. 93-98 (Wilson et al, eds., 12^(th) ed. 1991); Williams etal., J. of Medicinal Chem. 42:1481-1485 (1999).

“Somatic” pain, as described above, refers to a normal nerve response toa noxious stimulus such as injury or illness, e.g., trauma, burn,infection, inflammation, or disease process such as cancer, and includesboth cutaneous pain (e.g., skin, muscle or joint derived) and visceralpain (e.g., organ derived).

“Neuropathic” pain, as described above, refers to pain resulting frominjury to or chronic changes in peripheral and/or central sensorypathways, where the pain often occurs or persists without an obviousnoxious input.

The term “fever” as used herein refers to a patient's body temperaturewhich is higher than 100.4° F. (38° C.) measured rectally, higher than100° F. (37.8° C.) measured orally, or higher than 99° F. (37.2° C.)measured under the arm.

The term “inflammation” as used herein refers all categories ofinflammation, including localized manifestations and systemicinflammation (e.g., caused by infection with bacteria, fungi, orviruses, e.g., endotoxin, polysaccharide or viral proteins respectively,by inflammatory mediators, or as a consequence of auto-immunedisorders); inflammation that is categorized temporally, e.g., chronicinflammation and acute inflammation; inflammation that is categorized interms of its severity, e.g., mild, moderate, or severe; and inflammationthat is symptom or a result of a disease state or syndrome, e.g.,hepatitis type B liver inflammation. Inflammation, as used herein, canbe characterized at the “whole body” level as several localizedmanifestations (indices), including hemodynamic disorders (e.g.,hyperemia and edema), pain, temperature increment, and functionallesion. All indices may be observed in certain instances, although anyparticular indication is not necessarily always present. Concomitantcellular and molecular levels changes that characterize inflammation mayinclude leukocyte extravasation (a process involving adhesion ofleukocytes to the endothelium of the vessel wall and migration intotissue where they may phagocytose bacteria, viruses, and cell debris)and platelet aggregation (a mechanism whereby the spread of theinfection is prevented). Molecular level changes which characterizeinflammation may include activation of at least three plasma defensesystems (complement, kinin, and coagulation/fibrinolysis cascades) andsynthesis of cytokines and eicosanoids.

Inflammation as described above, refers to both cutaneous inflammation(e.g., skin, muscle or joint derived) and visceral inflammation (e.g.,organ derived). Cutaneous inflammation includes but is not limited toarthritis and dermatitis. “Arthritis” as used herein refers to, but isnot limited to, for example, rheumatoid arthritis, juvenile arthritis,psoriatic arthritis, Reiter's syndrome, ankylosing spondylitisarthritis, and gouty arthritis. “Dermatitis” as used herein is adisorder caused by inflammation to the skin including endogenous andcontact dermatitis such as, but not limited to, actinic dermatitis (orphotodermatitis), atopic dermatitis, chemical dermatitis, cosmeticdermatitis, dermatitis aestivalis, and seborrheic dermatitis.

The term “platelet-aggregation” as used herein refers to the process bywhich platelets bind to each other, e.g., at the site of vascularinjury, forming a platelet plug.

The phrase “side effects” encompasses unwanted and adverse effects of aprophylactic or therapeutic agent, e.g., aspirin. Adverse effects arealways unwanted, but unwanted effects are not necessarily adverse. Anadverse effect from a prophylactic or therapeutic agent might be harmfulor uncomfortable or risky. In certain embodiments, side effects from theadministration of aspirin, aspirin derivatives, other NSAIDs andanti-inflammatories include those which result from an inhibition ofTAFI, such as liver damage, stomach distress and bleeding complications,including but not limited to GI upset, occult bleeding, easy bruising,tinnitus, renal dysfunction and bronchospasm. Side effects fromadministration of SA include but are not limited to liver damage,stomach distress and bleeding complications, including but not limitedto GI upset, occult bleeding, easy bruising, tinnitus, renal dysfunctionand bronchospasm.

The term “prophylactic agent” means an agent capable of preventing orreducing the risks or incidence of a disease or disorder in a patient.In certain embodiments the prophylactic agent is capable of preventingor reducing the risks or incidence of thrombosis, hemorrhage, pain,inflammation, platelet aggregation, colon cancer or pancreatic cancer.

The phrase “prophylactically effective amount” refers to that amount ofthe prophylactic agent sufficient to result in the prevention orreduction in the risk or incidence of a disease or disorder, such as afever, a hemorrhagic, thrombotic, pain, inflammatory, plateletaggregation disease or disorder, colon cancer or pancreatic cancer.Further, a prophylactically effective amount with respect to aprophylactic agent of the invention means that amount of prophylacticagent alone, or in combination with other agents, that provides aprophylactic benefit in the prevention of the disease or disorder.

The term “therapeutic agent” means an agent capable of modifying,controlling, delaying or reversing a disease or disorder or amelioratingthe symptoms of a disease or disorder in a patient. In one embodiment,the therapeutic agent is one which is capable of modifying, controlling,delaying, reversing or ameliorating the symptoms of a fever, ahemorrhagic, thrombotic, pain, inflammatory, platelet aggregationdisease or disorder, colon cancer or pancreatic cancer.

The phrase “therapeutically effective amount” refers to that amount ofthe therapeutic agent sufficient to modify, control, reverse orameliorate the symptoms of the disease or disorder. In certainembodiments, the disease or disorder is a fever, a hemorrhagic orthrombotic disorder, pain, inflammatory disorder, platelet aggregationdisorder, colon cancer or pancreatic cancer. A therapeutically effectiveamount may refer to the amount of therapeutic agent sufficient to delay,minimize, reverse or ameliorate the symptoms of the disease or disorder.A therapeutically effective amount may also refer to the amount of thetherapeutic agent that provides a therapeutic benefit in the treatmentor management of the disease disorder. Further, a therapeuticallyeffective amount with respect to a therapeutic agent of the inventionmeans that amount of therapeutic agent alone, or in combination withother therapies, that provides a therapeutic benefit in the treatment ormanagement of the disease or disorder, including the amelioration ofsymptoms associated with the disease or disorder being treated. Atherapeutically effective amount may vary with a patient's age,condition and sex, as well as the extent of the disease in the patientand can be determined by one of skill in the art. The dosage may beadjusted by the individual physician or Veterinarian in the event of anycomplication.

A therapeutically effective amount typically will vary from about50-5,000 mg/day in one or more dose administrations daily, for one orseveral days, depending on the mode of administration and other factorsthat are apparent to one of skill in the art.

The phrase “a therapeutically effective amount” as used herein in thecontext of an analgesic refers to an amount of a compound effective toalleviate pain in vivo. The phrase “a therapeutically effective amount”as used herein in the context of a fever reducing compound refers to anamount of a compound effective to reduce the body temperature of apatient who is experiencing a fever. The phrase “a therapeuticallyeffective amount” as used herein in the context of an anti-inflammatoryrefers to an amount of a compound effective to alleviate inflammation invivo. The phrase “a therapeutically effective amount” as used herein inthe context of an anti-platelet aggregation compound refers to an amountof a compound effective to alleviate platelet aggregation in vivo. Thephrase “a therapeutically effective amount” as used herein in thecontext of a prophylactic colon cancer compound refers to an amount of acompound effective to prevent or reduce a patient's risk of colon cancerin vivo. The phrase “a therapeutically effective amount” as used hereinin the context of a prophylactic pancreatic cancer compound refers to anamount of a compound effective to prevent or reduce a patient's risk ofpancreatic cancer in vivo. The phrase “a therapeutically effectiveamount” as used herein in the context of a TAFI inhibitor refers to anamount of a compound effective to promote fibrinolysis in vivo.

The phrase “in combination” refers to the use of more than oneprophylactic and/or therapeutic agents against a disease or disorder,e.g., pain disorder and inflammation disorder. In combination may referto administration that is simultaneous, sequential and/or cyclical. Incertain embodiments, agents administered in combination may beadministered in the same pharmaceutical composition or separatepharmaceutical compositions.

The term “patient” refers to a mammal such as a non-primate (e.g., cows,pigs, horses, cats, dogs, mice, rats etc.) or a primate (e.g., monkeysand humans). Preferably the patient is a human.

The terms “manage”, “managing” and “management” refer to the beneficialeffects that a patient derives from a prophylactic or therapeutic agent,which does not result in a cure of the disorder. In certain embodiments,a patient is administered one or more prophylactic or therapeutic agentsto “manage” a disease or disorder, or symptoms associated with thedisease or disorder, so as to prevent the progression or worsening ofthe disease or disorder.

The terms “prevent”, “preventing” and “prevention” refer to theprevention of the recurrence, spread or onset of a disease or disorder,such as a pain, fever, inflammation, platelet aggregation disease ordisorder, colon cancer or pancreatic cancer, or symptoms associated withthe disease or disorder, in a patient resulting from the administrationof a prophylactic or therapeutic agent.

The terms “treat”, “treating” and “treatment” refer to the modification,control, reversal or amelioration of the symptoms of the disease ordisorder, such as a pain, fever, inflammation, platelet aggregationdisease or disorder, colon cancer or pancreatic cancer that results fromthe administration of one or more prophylactic or therapeutic agents. Incertain embodiments, such terms refer to the minimizing or delay of thehemorrhagic or thrombotic disease or disorder resulting from theadministration of one or more prophylactic or therapeutic agents to apatient with such a disease or disorder. In other embodiments, suchterms refer to the amelioration of one or more symptoms associated withan inflammatory disorder that results from the administration of one ormore prophylactic or therapeutic agents. In certain embodiments, suchterms refer to a reduction in the swelling of one or more joints, or areduction in the pain associated with an inflammatory disorder resultingfrom the administration of one or more prophylactic or therapeuticagents to a patient with such a disorder. In other embodiments, suchterms refer to a reduction in a patient of the symptoms of a fever,pain, platelet aggregation, colon cancer or pancreatic cancer thatresults from the administration of one or more prophylactic ortherapeutic agents.

It is noted that in this disclosure and particularly in the claims,terms such as “comprises”, “comprised”, “comprising” and the like canhave the meaning attributed to it in U.S. Patent law; e.g., they canmean “includes”, “included”, “including”, and the like; and that termssuch as “consisting essentially of” and “consists essentially of” havethe meaning ascribed to them in U.S. Patent law, i.e., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention. The singular forms “a”, “an” and “the” include pluralreferents, unless the content clearly dictates otherwise. Thus, forexample, reference to “an antigen” includes a mixture of two or moreantigens, reference to “an excipient” includes mixtures of two, or moreexcipients, etc.

Methods of Screening Compounds

In one embodiment, this invention provides assays for screening testcompounds for TAFIa inhibitory or non-inhibitory activity and for theability to treat, prevent or manage pain, fever, inflammation, plateletaggregation, colon cancer, pancreatic cancer, thrombotic andfibrinolytic diseases and disorders, i.e., for other aspirin activities.

Test Compounds

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including but not limited to biological libraries, spatiallyaddressable parallel solid phase or solution phase libraries, syntheticlibrary methods requiring deconvolution, the “one-bead one-compound”library method, and synthetic library methods using affinitychromatography selection. The biological library approach is used withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, K. S. (1997) Anticancer Drug Des. 12:145.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In particular, any known analgesic, antipyretic, anti-inflammatory,anti-platelet aggregation, colon cancer prophylactic, pancreatic cancerprophylactic, fibrinolytic or thrombotic agent may be screened by themethods of the invention including but not limited to aspirin,ibuprofen, acetominophen, Nabumetone (RELAFEN®), salsalate, Etodac(LODINE®), Ibuprofen, Aspirin, Diclofenac (VOLTAREN®, CATAFLAM®),Sulindac (CLINORIL®), Diflunisal (DOLOBID®), Naproxen (NAPROSYN®,ALLEVE®), Indomethacin (INDOCIN®), Tolmetin sodium (TOLECTIN®),Fenoprofen calcium (NALFON PULVULES®), Ketoprofen (ORUDIS®, ORUVAIL®),Piroxicam (FELDENE®), Flubiprofen (ANSAID®), Meclofenamate sodium(MECLOMEN®), Ketorolac tromethamine (TORADOL®), Oxaprozin (DAYPRO®),COX-1 inhibitors, and COX-2 inhibitors including but not limited toCELEBREX® and Vioxx®, and metabolites and derivatives thereof.

Compounds identified by the methods of the invention may then bederivatized using methods well known to one of skill in the art.Additionally, compounds may be designed through rational drug designprocedures known in the art. In a particular embodiment, compounds whichmimic the TAFIa inhibitory activity of salicylic acid, salicyluric acidor gentisic acid may be designed through rational drug designprocedures. These procedures include, but are not limited to X-raycrystallography, computer assisted modeling and analog synthesis.

In another embodiment, antibodies are screened by the methods of theinvention. In particular, antibodies may be screened for analgesic,antipyretic, anti-inflammatory, anti-platelet aggregation, colon cancerprophylactic and/or colon cancer prophylactic activities.

In one embodiment, monoclonal antibodies can be screened. In anotherembodiment, libraries of antibodies can be screened. In particular,phage display methods may be utilized wherein functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of lymphoid tissues). The DNAencoding the VH and VL domains are recombined together with an scFvlinker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 orpComb 3 HSS). The vector is electroporated in E. coli and the E. coli isinfected with helper phage. Phage used in these methods are typicallyfilamentous phage including fd and M13 and the VH and VL domains areusually recombinantly fused to either the phage gene III or gene VIII.Phage expressing an antigen binding domain that binds to a particularantigen (e.g., a polypeptide) can be selected or identified with antigen(e.g., TAFI), e.g., using labeled antigen or antigen bound or capturedto a solid surface or bead. Alternatively, phage can be assayed in theassay methods of the invention. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough etal., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCTApplication No. PCT/GB91/O1134; PCT Publication Nos. WO 90/02809, WO91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including subjectian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)2 fragments can also be employed using methods knownin the art such as those disclosed in PCT Publication No. WO 92/22324;Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995,AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043.

Methods of Screening for Compounds to Treat, Prevent or Manage Pain,Fever, Inflammation, Platelet-Aggregation, Colon Cancer or PancreaticCancer

One aspect of the invention provides a method of screening for compoundswhich retain at least one aspirin activity, e.g. COX-2 activity, but donot inhibit TAFIa activity and/or TAFI activation. In preferredembodiments, the test compounds are screened for at least one otheraspirin activity such as ability to treat, prevent or manage pain,fever, inflammation, platelet aggregation, colon cancer, pancreaticcancer, COX-1 and/or COX-2 inhibition.

In one embodiment, the invention provides a method of screeningcompounds for at least one aspirin activity including, but not limitedto, analgesia, fever reduction, anti-inflammation, anti-plateletaggregation, colon cancer prophylaxis and pancreatic cancer prophylaxis,and for non-inhibition of TAFI. The method comprises the steps of (a)exposing TAFI to a test compound, (b) determining whether the testcompound modulates TAFIa activity and/or TAFI activation and (c)determining whether at least one aspirin activity is retained by thetest compound. In a particular embodiment, a method of screening foranalgesic activity and non-inhibition of TAFI is provided. In aparticular embodiment, a method of screening for antipyretic activityand non-inhibition of TAFI is provided. In another embodiment, a methodof screening for anti-inflammatory activity and non-inhibition of TAFIis provided. In another embodiment, a method of screening foranti-platelet aggregation and non-inhibition of TAFI is provided. Inanother embodiment, a method of screening for colon cancer prophylaxisand non-inhibition of TAFI is provided. In a particular embodiment, amethod of screening for pancreatic cancer prophylaxis and non-inhibitionof TAFI is provided.

A second aspect of this invention relates to screening compounds forTAFIa inhibitory activity, comprising (a) exposing TAFI to a testcompound; and (b) determining whether the test compound inhibits TAFIaactivity and/or TAFI activation. In a particular embodiment, the methodfurther comprises comparing the TAFI inhibitory activity of the testcompound to that of a compound selected from the group consisting ofacetylsalicylic acid, salicylic acid, salicyluric acid or gentisic acid.In a particular embodiment, the invention relates to a method ofscreening compounds for anti-TAFIa/TAFI activity but not aspirinactivity.

The analgesic, antipyretic, anti-inflammatory, anti-plateletaggregation, colon cancer prophylactic or pancreatic cancer prophylacticactivity of a test compound can be assessed using a variety of in vitroand in vivo assays known to one of skill in the art.

COX-1 and COX-2 activity may be measured by various methods well knownin the art. In particular, COX-1 activity may be assessed by determiningplatelet aggregation and thromboxane (TX) production. COX-2 activity maybe measured by assessing prostaglandin 2 (PGE2) production inlipopolysaccharide (LPS)-stimulated monocytes. In preferred embodiments,the in vitro studies are conducted by taking whole blood from healthypatients and exposing it to the test compound. In preferred embodiments,ex vivo studies are conducted using whole blood from patients exposed todrugs. In a particularly preferred embodiment, the ex vivo whole bloodassay methods described in Patrignani et al., 1994, J. Pharmacol. Exp.Ther. 271:1705-1712 are used. Other preferred assays for determiningCOX-1 and COX-2 inhibition include but are not limited to thosedescribed in Vane et al., 1998, Am. J. Med., 104:2 S-8S; Fenner, H.,1997, Semin. Arthritis Rheum. 26:28-33; Cryer, B. and Feldman, M., Am.J. Med. 104:413-421.

The analgesic activity of a compound of the invention may be evaluated,for example, in vivo by the formalin test. In the formalin test, miceare administered an IP dose of the test compound or a control (i.e., aknown analgesic or a non-analgesic) compound. Thirty minutes later, aformalin solution is injected into the plantar surface of the right hindpaw. For thirty minutes immediately following the injection, the miceare observed and the time spent licking the paw (a response to pain) ismeasured using a timer. The time spent licking the right hind paw by acontrol mouse is compared to that spent licking by a mouse that wasadministered the test compound.

Additionally, the analgesic effect of a compound may be evaluated invivo by the hotplate test. In the hotplate test, mice are administered adose of the test compound or control compound. One hour later, the miceare placed on a metal surface heated to 55° C. When the mouse licks itshind paw or after 30 seconds, it is removed from the surface, and thelatency to the lick is measured. The time difference between the treatedand control mice is then compared.

In one embodiment of the invention, pain is alleviated by at least 400%,at least 350-400%, at least 300-350%, at least 275-300%, at least250-275%, at least 225-250%, at least 200-225%, at least 150-200%, atleast 100-150%, at least 50-100% or at least 1-50% as compared toequivalent amounts of a known analgesic. Known analgesics include butare not limited to aspirin, VIOXX®, CELEBREX®, ibuprofen, acetaminophen,codeine and other NSAIDs. In preferred embodiments, pain is alleviatedby more than 200%, as compared to equivalent amounts of a knownanalgesic.

The anti-inflammatory activity of therapies used in accordance with thepresent invention can be determined by using various experimental animalmodels of inflammatory arthritis known in the art and described inCrofford L. J. and Wilder, R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al., (eds.), Chapter 30 (Lee and Febiger,1993). The following assays are provided as examples and not bylimitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis rat,rabbit and hamster models, all described in Crofford L. J. and Wilder,R. L., “Arthritis and Autoimmunity in Animals”. In Arthritis and AlliedConditions: A Textbook of Rheumatology, McCarty et al., (eds.), Chapter30 (Lee and Febiger, 1993).

The anti-inflammatory activity of the therapy may be determined using acarrageenan-induced arthritis rat model. Carrageenan-induced arthritishas previously been used in rabbit, dog and pig studies of chronicarthritis or inflammation. Quantitative histomorphometric assessment isused to determine therapeutic efficacy. The methods of using suchcarrageenan-induced arthritis model are described in Hansra P., et al.,2000, Inflammation 24(2):141-155.

Additionally, the anti-inflammatory activity of a test compound may beevaluated by the pleural exudation assay, the formaldehyde inducedarthritis assay or the cotton pellet implantation assay as fullydescribed in Spector, W. G., 1956, J. Path. Bact. 12:367-380, Brownlee,G., 1950, Lancet 1: 157-159, and Meier, R., Schuler W., and Desaulles,P., 1950, Experintia 6:469, respectively. Briefly, experimental pleurisyis produced by injecting a rat with 0.1 mL of turpentine into the rightpleural space under light ether anaesthesia. The test anti-inflammatorycompound may be injected intraperitoneally in graded doses 1 hour beforeturpentine injection. The rats are decapitated and pleural exudate iscollected half and hour after turpentine treatment. The volume ofexudate is measured and compared to that of rats that have beenadministered control substances or known anti-inflammatories.

In the formaldehyde induced arthritis assay, 0.1 mL of 2% (v/v)formaldehyde is injected subcutaneously under the plantar aponeurosis ineach foot of the rat on the first and third days. The testanti-inflammatory compound may be given intraperitoneally once daily for10 days in graded doses. Day-to-day changes in the inflammatory reactionare assessed by measuring the linear cross-section immediately below theankle joint with a micrometer screw gauge. The diameter of ankle is thencompared to that of rats that have been administered control substancesor known anti-inflammatories.

In the cotton implantation assay, pellets of surgical cotton weighing9.0±1 mg are sterilized in an air oven for 2 hours and implanted in boththe axillae and groins under ether anaesthesia. The testanti-inflammatory compound is given intraperitoneally in graded dosesdaily for 6 days. The pellets are dissected out on the 7^(th) day underlight ether anaesthesia. They are kept separately in small glass vials,dried for 2 hours at temperature of 150 C. and weighed after cooling.The weight of granulation tissue is then compared to that of rats thathave been administered control substances or known anti-inflammatories.

The anti-inflammatory cyclo-oxygenase inhibitory activity of a testcompound may be screened by the ovine ureteral motility assay asdescribed in Bhargava, K. P. and Thulesius, O., 1987, Gen. Pharmacol.18(3):337-40. Briefly, an isolated sheep ureteral ring is suspended inan organ bath. The ureteral ring will exhibit rhythmic contractionswhich are dose-dependently inhibited by indomethacin and revived byprostaglandin E2 or F2 alpha. Test compounds are tested on the isolatedureteral ring model to determine the time elapsing from application oftest compound to complete inhibition of ureteral contraction, i.e.,“stop time.” Stop times are then compared to stop times in the absenceof the compound or in presence of a control substance or a knowncyclo-oxygenase inhibitor.

The invention also provides methods of screening anti-plateletaggregation in vivo and in a patient. For example, platelet aggregationmay be evaluated using the procedure described in Aguejouf et al 2000,Thrombosis Research 99: 595-602. In brief, a rat is exposed to a testcompound, administered anesthesia and then a median laparotomy isperformed. The intestinal loop is placed on a microscope table andvascular lesions are induced by a laser (e.g. Stabilite 2016, SpectraPhysics, France). The procedure lasts 10 minutes and is repeated foreach rat. Platelet aggregation is then evaluated according to themethods described in Cardinal, D. and Flower, R., 1980, J.Pharmacological Methods 135-8.

In one embodiment of the invention, platelet aggregation is reduced byat least 91-100%, 81-90%, 71-80%, 61-70%, 51-60%, 41-50%, 31-40%,21-30%, 11-20% or 1-10% as compared to equivalent amounts of a knownanti-platelet aggregation compound, including but not limited toaspirin. In preferred embodiments, platelet aggregation is reduced bymore than 100% as compared to equivalent amounts of a knownanti-platelet aggregation compound.

Test compounds which retain the desired amount of at least one aspirinactivity can then be tested for modulation of TAFIa activity. In apreferred embodiment, the test compounds are tested for non-inhibitionof TAFIa activity. In a particular embodiment, test compounds arescreened for modulation of TAFIa carboxypeptidase activity. In apreferred embodiment, test compounds are screened for the ability toblock, inhibit or retard the cleavage of fibrin or other substrates byTAFIa. In one embodiment, TAFIa carboxypeptidase activity is measuredusing a fluorometric in vitro assay. In particularly preferredembodiments, TAFIa carboxypeptidase activity may be measured accordingto the exemplary teachings set forth in U.S. patent application Ser. No.10/116,095, filed Apr. 4, 2002 and U.S. Provisional Patent ApplicationNo. 60/406,756 entitled “Diagnostic Assay for Thrombin-ActivatableFibrinolysis Inhibitor (TAFI)”, by Greenfield and An. In a particularlypreferred embodiment, levels of TAFIa may be measured using the ELISAprocedure described therein. In certain embodiments, modulation of TAFIaactivity may be measured by determining whether TAFIa enzymatic activityis changed in step (a) relative to TAFIa exposed to aspirin, salicylicacid, salicyluric acid or gentisic acid.

TAFIa activity may by measured by the clot lysis assay. See Reed, G., etal., 1990, Proc. Nat'l Acad. of Sci. USA 87: 1114-1118 as described inU.S. Pat. No. 6,114,506, issued Sep. 5, 2000.

TAFIa activity may also be measured by the fibrinogen assay. SeeRampling, M. W. and Gaffney, P. J., 1976, Clin. Chim, Acta. 67: 43-53,which is hereby incorporated be reference in its entirety. See U.S. Pat.No. 6,114,506, issued Sep. 5, 2000.

In a preferred embodiment, TAFIa is preincubated with the test compoundfor about 1 hour. In various embodiments, TAFIa is preincubated with thetest compound for about 30 minutes, less than 30 minutes, at least 30minutes, less than 1 hour, at least 1 hour, about 2-3 hours, about 4-5hours, about 6-8 hours, about 8-10 hours, about 10-12 hours, about 12-16hours, about 16-20 hours, about 24 hours, at least 24 hours or less than24 hours. In one embodiment, TAFIa is preincubated with the testcompound at around room temperature. In another embodiment, TAFIa ispreincubated with the test compound at around 37° C.

In another embodiment, modulation of TAFIa activity is measured bydetermining whether TAFI proenzyme to TAFIa conversion is changed instep (a) relative to TAFIa exposed to aspirin, salicylic acid,salicyluric acid or gentisic acid. In a particular embodiment, TAFIproenzyme to TAFIa conversion by thrombin/thrombomodulin in the presenceof a test compound is measured. The levels of TAFI proenzyme can becompared to levels of TAFIa in aspirin and aspirin metabolite exposedsamples. In a preferred embodiment, TAFIa is preincubated with the testcompound for about 1 hour.

In preferred embodiments, the test compound inhibits TAFI activityand/or activation by at least 100%, about 100%, about 50-100%, about25-50%, about 1-25%, preferably about 1-15%, or most preferably about1-5%, as compared to aspirin, salicylic acid, salicyluric acid orgentisic acid. Alternatively, the test compound promotes TAFI activityand/or activation.

Methods of Screening for Compounds to Treat, Prevent or ManageFibrinolysis or Thrombosis

The instant invention provides methods of screening compounds for TAFIainhibitors. Such compounds are useful for the treatment of thromboticdiseases including but not limited to angina pectoris, chronic stableangina pectoris, heart attack, stroke, thromboembolic disease,myocaridal infarction (MI), acute myocardial infarction (AMI), recurrentmyocardial infarction, ischemic attack, transient ischemic attack (TIA),deep vein thrombosis, acute ischemic stroke, massive pulmonary embolism,disseminated intravascular coagulation (DIC), anti-phospholipidsyndrome, familial thrombophilia, sepsis, arthritis, fulminanthepatitis, thrombosis, hemophilia and Von Willebrand disease.

In one embodiment, modulation of TAFIa activity is measured bydetermining whether TAFIa enzymatic activity is changed in step (a)relative to TAFIa exposed to aspirin, salicylic acid, salicyluric acidor gentisic acid. In a particular embodiment, test compounds arescreened for those which block, inhibit or retard the cleavage of fibrinor other substrates by TAFIa. In a particular embodiment, inhibition ofTAFIa carboxypeptidase activity is measured. In a preferred embodiment,TAFIa carboxypeptidase activity is measured using a fluorometric invitro assay. In particularly preferred embodiments, TAFIacarboxypeptidase activity may be measured according to the exemplaryteachings set forth in U.S. patent application Ser. No. 10/116,095,filed Apr. 4, 2002 and U.S. Provisional Patent Application No.60/406,756, entitled “Diagnostic Assay for Thrombin-ActivatableFibrinolysis Inhibitor (TAFI)”, by Greenfield and An. In a particularlypreferred embodiment, levels of TAFIa may be measured using the ELISAprocedure described therein.

In a preferred embodiment, TAFIa is preincubated with the test compoundfor about 1 hour. In various embodiments, TAFIa is preincubated with thetest-compound for less than 1 hour, at least 1 hour, about 30 minutes;about 2-3 hours, about 4-5 hours, about 6-8 hours, about 8-10 hours,about 10-12 hours, about 12-16 hours, about 16-20 hours, about 24 hours,at least 24 hours or less than 24 hours. In one embodiment, TAFIa ispreincubated with the test compound at room temperature. In anotherembodiment, TAFIa is preincubated with the test compound at 37° C.

In another embodiment, modulation of TAFIa activity is measured bydetermining whether TAFI proenzyme to TAFIa conversion is changed instep (a) relative to TAFIa exposed to aspirin, salicylic acid,salicyluric acid or gentisic acid. In a particular embodiment, TAFIproenzyme to TAFIa conversion by thrombin/thrombomodulin in the presenceof a test compound is measured. The levels of TAFI proenzyme can becompared to levels of TAFIa in aspirin and aspirin metabolite exposedsamples. In a preferred embodiment, TAFIa is preincubated with the testcompound for about 1 hour. In another embodiment, TAFIa is preincubatedwith the test compound for about 30 minutes.

In certain embodiments, the test compound reduces TAFIa activity by atleast at least 400%, at least 375-400%, at least 350-375%, at least325-350%, at least 300-325%, at least 275-300%, at least 250-275%, atleast 225-250%, at least 200-225%, at least 150-200% at least 100-150%,at least 50-100% or at least 1-50% as compared to TAFIa activity in theabsence of the test compound. In certain embodiments, the modulation ofTAFIa activity in the presence of the test compound is compared to TAFIaactivity in the presence of equivalent amounts of known TAFIa inhibitorssuch as aspirin, salicylic acid, salicyluric acid or gentisic acid.

Pharmaceutical Formulations and Applications

The compounds identified by the methods of the invention are useful inthe methods of treatment, prevention and management of pain, fever,inflammation, platelet aggregation, fibrinolysis, thrombosis, coloncancer, pancreatic cancer and the diseases and disorders associatedtherewith. Additionally, the compounds identified by the screeningmethods of the present invention can used in prophylactic andtherapeutic methods, administered to the patient population, used incombination therapy, formulated in pharmaceutical compositions, andadministered by the methods and in the dosages set forth in thefollowing sections of the specification.

Prophylactic and Therapeutic Methods Using the Compounds of theInvention

The present invention encompasses methods for treating, preventing, ormanaging any disease or disorder which can be treated, prevented ormanaged by aspirin therapy, other NSAIDs or anti inflammatories. Thepresent invention also encompasses methods of eliminating at least oneaspirin associated adverse side effect that can be prevented or managedby modulating TAFIa activity.

Hemorrhagic and thrombotic diseases and side effects occur, in part,because the normal balance between the coagulation and fibrinolyticcascades has been affected, altered or shifted. The methods of thepresent invention allow the balance of the cascades to be shifted byindividually increasing or decreasing one of the respective cascadeswithout affecting the other, e.g., by providing TAFIa activity tocompensate for the TAFIa inhibitory activity of aspirin.

Another aspect of the present invention is directed to a method oftreating, preventing or managing bleeding side-effects associated withthe administration of aspirin or any other anti-coagulant, such astissue plasminogen activator (t-PA), urokinase plasminogen activator(u-PA), streptokinase, staphylokinase, plasminogen or analogs thereof,heparin or low molecular weight heparin, or an anti-inflammatory, suchas hydrocoftisone, sodium salicylate, oxyphenbutazone, indomethacin orcyproheptadine, or any other anti-cancer drug, such as methotrexate,taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine,cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,mitomycin, dacarbazine, procarbizine, etoposides, campathecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin,5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin,levamisole, irinotecan, estramustine, etoposide, nitrosoureas such ascarmustine and lomustine, vinca alkaloids, platinum compounds,mitomycin, gemcitabine, hexamethylmelamine and topotecan, comprisingadministering a therapeutically or prophylactically effective amount ofa compound which maintains other aspirin activities, e.g., COX-2activity, but does not inhibit TAFIa activity or TAFI activation. In oneembodiment, the aspirin activity is analgesia. In another embodiment,the aspirin activity is anti-pyretic activity. In another embodiment,the aspirin activity is anti-inflammation activity. In anotherembodiment, the aspirin activity is anti-platelet aggregation activity.In another embodiment, the aspirin activity is colon cancer prophylaxis.In another embodiment, the aspirin activity is pancreatic cancerprophylaxis.

Another aspect of the present invention is directed to a method oftreating, preventing or managing bleeding side-effects associated withthe administration of aspirin or any other anti-coagulant, such as,tissue plasminogen activator (t-PA), urokinase plasminogen activator(u-PA), streptokinase, staphylokinase, plasminogen or analogs thereof,heparin or low molecular weight heparin, or an anti-inflammatory, suchas hydrocortisone, sodium salicylate, oxyphenbutazone, indomethacin orcyproheptadine, or an anti-cancer drug, such as methotrexate, taxol,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,dacarbazine, procarbizine, etoposides, campathecins, bleomycin,doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin,mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine,paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil,taxanes such as docetaxel and paclitaxel, leucovorin, levamisole,irinotecan, estramustine, etoposide, nitrosoureas such as carmustine andlomustine, vinca alkaloids, platinum compounds, mitomycin, gemcitabine,hexamethylmelamine and topotecan, comprising administering atherapeutically or prophylactically effective amount of stabilizedTAFIa. In a particular embodiment, stabilized TAFIa is administeredprior to the administration of aspirin or other therapeutic agent. Inanother particular embodiment, stabilized TAFIa is administeredsubsequent to the administration of aspirin or other therapeutic agent.In another embodiment, stabilized TAFIa is administered concurrentlywith aspirin or other therapeutic agent. In another embodiment,stabilized TAFIa is administered simultaneously, e.g., in the samecomposition, as the administration of aspirin or other therapeuticagent.

The term “concurrently” is not limited to the administration ofprophylactic or therapeutic agents at exactly the same time, but ratherit is meant that the agents are administered within a time interval suchthat the two agents can work together. For example, one agent may beadministered one time per week in combination with another agent that isone time every two weeks or one time every three weeks.

The therapeutic or prophylactic: agents used in combination may beadministered by the same or different routes of administration, e.g.,oral and parenteral.

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered in combination to a patient.Cycling therapy involves the administration of a first agent for aperiod of time, followed by administration of a second agent and/orthird agent for a period of time and repeating this sequentialadministration. Cycling therapy reduces the development of resistance toone or more of the therapies, avoids or reduces the side effects of oneof the therapies, and/or improves the efficacy of the treatment.

Another aspect of the present invention is directed to a method oftreating, preventing or managing a thrombotic disease or disordercomprising administering a therapeutically or prophylactically effectiveamount of a compound of the invention, e.g., as identified by thescreening methods of the invention, or a pharmaceutical compositionthereof, to a patient. In certain embodiments, the thrombotic diseasesor disorders include, but are not limited to, heart attack, stroke,thromboembolic disease, acute myocardial infarction (AMI), deep veinthrombosis, acute ischemic stroke, massive pulmonary embolism,disseminated intravascular coagulation (DIC), familial thrombophilia,sepsis, arthritis, fulminant hepatitis and thrombosis. In preferredembodiments, the inhibitor of TAFIa is salicylic acid.

Suitable regimens can be selected by one skilled in the art byconsidering such factors and by following, for example, dosages reportedin the literature and recommended in the Physician's Desk Reference(56^(th) ed., 2002).

Patient Population

The invention provides methods of treating a patient suffering frompain, fever, colon cancer, pancreatic cancer, coronary artery disease,inflammatory disease, platelet aggregation disease or fibrinolyticdisease which can be treated, prevented or managed by administering tothe patient a therapeutically or prophylactically effective amount of acompound identified by the screening methods of the present invention,or a pharmaceutical composition thereof.

The invention also provides methods of treating a patient suffering froma thrombotic or hemorrhagic disorder, or any other disease or disorderassociated with aberrant levels of TAFIa or which can be treated,prevented or managed by decreasing TAFIa activity, by administrating toa patient a therapeutically or prophylactically effective amount of aninhibitor of TAFIa identified by a screening method of the invention, orpharmaceutical composition thereof.

The patient is preferably a mammal such as a non-primate (e.g., cows,pigs, horses, cats, dogs, mice, rats etc.) or a primate (e.g., monkeys,such as cynomolgus monkeys, and humans). In a preferred embodiment, thepatient is a human.

The invention also encompasses methods for treating patients that arereceiving or have received any other treatment useful for the preventionof pain, fever, inflammation, platelet aggregation, colon cancer,pancreatic-cancer, thrombotic or hemorrhagic diseases or disorders. Inanother embodiment, the patient has never received any other treatmentuseful for the prevention of pain, fever, inflammation, plateletaggregation, colon cancer, pancreatic cancer, thrombotic or hemorrhagicdiseases or disorders.

Patients can be treated for somatic pain, neuropathic pain, localizedinflammation, systemic inflammation, arthritis or dermatitis using themethods of the invention.

Using the methods of the present invention, patients can be treated forthe prevention, treatment or management of hemorrhagic or thromboticdiseases or disorders, including, but not limited to, heart attack,stroke, thromboembolic disease, hemophilia, von Willebrand disease(VWD), acute myocardial infarction (AMI), deep vein, thrombosis, acuteischemic stroke, massive pulmonary embolism, disseminated intravascularcoagulation (DIC), familial thrombophilia, sepsis, arthritis, Fulminanthepatitis, Henoch-Schonlein purpura, hemostasis and thrombosis, or anydisease or disorder characterized by excessive clotting. In oneembodiment, patients can be treated for hemorrhagic diseases ordisorders which include, but are not limited to, hemophilia A,hemophilia B, autoimmune haemolytic anaemia, collagen diseases, vonWillebrand disease (VWD), Henoch-Schonlein purpura, acute-generalizedwidespread bleeding, primary hyperfibrinolysis, hepatosplenicschistosomiasis, factor deficiencies and hemostasis.

In another embodiment, the methods of the present invention are usefulfor the amelioration of symptoms associated with hemorrhagic orthrombotic diseases or disorders. The methods and compositions of theinvention can be used with one or more conventional or experimentaltherapies that are used to prevent, treat or manage hemorrhagic orthrombotic diseases or disorders.

In one embodiment, the invention provides methods for treating,preventing, or managing thrombotic diseases or disorders byadministrating a therapeutically or prophylactically effective amount ofa TAFIa inhibitor identified by a method of the invention, or apharmaceutical composition thereof to a patient currently undergoingalternative treatment for a thrombotic disease or disorder.

In another embodiment, the invention provides methods for treating,preventing, or managing thrombotic diseases or disorders by:administrating a therapeutically or prophylactically effective amount ofa TAFIa inhibitor identified by a method of the invention, or apharmaceutical composition thereof to a patient who has previouslyundergone treatment for a thrombotic disease or disorder.

In another embodiment, the invention provides methods for treating,preventing, or managing thrombotic diseases or disorders byadministrating a therapeutically or prophylactically effective amount ofa TAFIa inhibitor identified by a method of the invention, or apharmaceutical composition thereof to a patient who has never undergonetreatment for a thrombotic disease or disorder.

Combination Therapies

According to the invention, therapy by administration of a compound ofthe invention, or a pharmaceutical composition thereof is combined withthe administration of one or more therapeutic or prophylactic agentssuch as, but not limited to, analgesics, antipyretics,anti-inflammatories, anti-platelet aggregation compounds, colon cancerprophylactics, pancreatic cancer prophylactics and procoagulant agents.When used in combination with other prophylactic and/or therapeuticagents, the compounds identified by the screening methods of the presentinvention, or pharmaceutical compositions thereof, can be administeredprior to, subsequent to or concurrently with the other therapeutic orprophylactic agents.

In one embodiment, the therapeutic or prophylactic agent is useful forthe treatment, prevention or management of at least one of thefollowing: pain, fever, inflammation, platelet aggregation, fibrinolysisor hemorrhage, colon cancer or pancreatic cancer. In other embodiments,the therapeutic or prophylactic agent is useful for the treatment,prevention or management of at least 2-3 of the following: pain, fever,inflammation, platelet aggregation, fibrinolysis or hemorrhage, coloncancer or pancreatic cancer. In other embodiments, the therapeutic orprophylactic agent is useful for the treatment, prevention or managementof at least 4-5 of the following: pain, fever, inflammation, plateletaggregation, fibrinolysis or hemorrhage, colon cancer or pancreaticcancer.

In another embodiment, the inhibitor of TAFIa, or pharmaceuticalcomposition thereof, can be administered in combination with anotherprophylactic or therapeutic agent. When used in combination with otherprophylactic and/or therapeutic agents, the inhibitor of TAFIa, orpharmaceutical composition thereof can be administered prior to,subsequent to or concurrently with one or more other therapeutic orprophylactic agents. In one embodiment, the inhibitor of TAFIa, orpharmaceutical composition thereof is administered concurrently with oneor more therapeutic or prophylactic agents, e.g., in the samepharmaceutical composition. In another embodiment, the inhibitor ofTAFIa, or pharmaceutical composition thereof is administeredconcurrently with one or more other therapeutic or prophylactic agentsin separate pharmaceutical compositions. In certain embodiments, theother therapeutic or prophylactic agent is useful for the treatment,prevention or management of a thrombotic disorder. In other embodiments,the therapeutic or prophylactic agent is not useful for the treatment,prevention or management of a thrombotic disorder. In certainembodiments, the therapeutic or prophylactic agent includes, but is notlimited to aspirin, t-PA, heparin, Factor VII, Factor VIII, Factor IX,amino caproic acid, activated protein C, thrombin, fibrinogen,cryofractionalized plasma, actothrombin, low molecular weight heparin,hirudin, plasminogen, streptokinase, staphylokinase, urokinase orhirulong.

Examples of analgesics and anti-inflammatories include but are notlimited to aspirin, ibuprofen; acetominophen, methotrexate, Nabumetone(RELAFEN®), salsalate, Etodac (LODINE®), Ibuprofen, Aspirin, Diclofenac(VOLTAREN®), CATAFLAM®), Sulindac (CLINORIL®), Diflunisal (DOLOBID®),Naproxen (NAPROSYN®, ALLEVE®), Indomethacin (INDOCIN®), Tolmetin sodium(TOLECTIN®), Fenoprofen calcium (NALFON PULVULES®), Ketoprofen (ORUDIS®,ORUVAIL®), Piroxicam (FELEDENE®), Flubiprofen (ANSAID®), Meclofenamatesodium (MECLOMEN®), Ketorolac tromethamine (TORADOL®), Oxaprozin(DAYPRO®), COX-1 inhibitors, and COX-2 inhibitors including but notlimited to CELEBREX® and VIOXX®. Additional anti-inflammatories includebut are not limited to beta-agonists, anticholinergic agents, methylxanthines and corticosteroids (e.g., prednisone and hydrocortisone).

Examples of anti-coagulant agents include, but are not limited to, t-PA(or analogs thereof), heparin, low molecular weight heparin, aspirin,actothrombin, hirudin, plasminogen, streptokinase, staphylokinase,urokinase or hirulong.

Examples of anti-cancer drugs include, but are not limited tomethotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea,cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin,carboplatin, mitomycin, dacarbazine, procarbizine, etoposides,campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin,epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel,leucovorin, levamisole, irinotecan, estrarnustine, etoposide,nitrosoureas such as carmustine and lomustine, vinca alkaloids, platinumcompounds, mitomycin, gemcitabine, hexamethylmelamine and topotecan.

In one embodiment, a compound of the invention, or a pharmaceuticalcomposition thereof is administered in combination with surgery. Theadministration can be either prior to surgery, after surgery orconcomitant with surgery. Examples of such surgeries include, but arenot limited to organ transplant surgery, cardiopulmonary bypass surgery,coronary angioplasty, open heart surgery, heart valve surgery or removalof atherosclerotic plaques. In preferred embodiments, the inhibitor ofTAFIa is salicylic acid.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention comprise aprophylactically or therapeutically effective amount of a compound ofthe invention, alone or in combination with another prophylactic ortherapeutic agent, and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of a compound of the invention, and apharmaceutically acceptable carrier.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound of the invention is administered. Suchpharmaceutical vehicles can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical vehicles can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea, and the like. In addition,auxiliary, stabilizing, thickening, lubricating and coloring agents maybe used. When administered to a patient, the compounds of the inventionand pharmaceutically acceptable vehicles are preferably sterile. Wateris a preferred vehicle when the compound of the invention isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid vehicles, particularlyfor injectable solutions. Suitable pharmaceutical vehicles also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propyleneglycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable vehicle is acapsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical vehicles are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

In a preferred embodiment, the compounds of the invention are formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compounds of the invention for intravenous administration are solutionsin sterile isotonic aqueous buffer. Where necessary, the compositionsmay also include a solubilizing agent. Compositions for intravenousadministration may optionally include a local anesthetic such asLidocaine (lignocaine) to ease pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecompound of the invention is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the compound of theinvention is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

Compositions for oral delivery may be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions maycontain one or more optional agents, for example, sweetening agents suchas fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions may be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds ofthe invention. In these later platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving compound, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time delay material such as glycerol monostearate orglycerol stearate may also be used. Oral compositions can includestandard vehicles such as mannitol, lactose, starch, magnesium stearate,sodium saccharin, cellulose, magnesium carbonate, etc. Such vehicles arepreferably of pharmaceutical grade.

Further, the effect of the compounds of this invention may be delayed orprolonged by proper formulation. For example, a slowly soluble pellet ofthe compound may be prepared and incorporated in a tablet or capsule.The technique may be improved by making pellets of several differentdissolution rates and filling capsules with a mixture of the pellets.Tablets or capsules may be coated with a film which resists dissolutionfor a predictable period of time. Even the parenteral preparations maybe made long-acting, by dissolving or suspending the compound in oily oremulsified vehicles which allow it to disperse only slowly in the serum.

Routes of Administration

Methods of administering a compound of the invention, or apharmaceutical composition thereof include, but are not limited to,parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural, and mucosal(e.g., intranasal, rectal, vaginal, sublingual, buccal or oral routes).In a specific embodiment, a compound of the invention, or apharmaceutical composition thereof is administered intramuscularly,intravenously, or subcutaneously. The compound of the invention, or apharmaceutical composition thereof may also be administered by infusionor bolus injection and may be administered together with othertherapeutic or prophylactic agents. Administration can be local orsystemic. The compound of the invention, or pharmaceutical compositionthereof may also be administered by inhalation or insufflation (eitherthrough the mouth or the nose). In a preferred embodiment, local orsystemic parenteral administration is used.

In specific embodiments, it may be desirable to administer the compoundof the invention, or a pharmaceutical composition thereof, locally tothe area in need of treatment. This may be achieved, for example, andnot by way of limitation, by local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as silastic membranes, orfibers. In one embodiment, administration can be by direct injection atthe site (or former site) of an atherosclerotic plaque tissue.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the compound of the invention, or a pharmaceuticalcomposition thereof can be formulated as a suppository, with traditionalbinders and vehicles such as triglycerides.

In another embodiment, the compound of the invention, or apharmaceutical composition thereof, can be delivered in a vesicle, inparticular a liposome (see Langer, 1990, Science 249:1527-1533; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez -Berestein, ibid., pp. 317-327; see generally ibid.).

In yet another embodiment, the compound of the invention, or apharmaceutical composition thereof, can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald etal., 1980, Surgery 88:507 Saudek et al., 1989, N. Engl. J. Med.321:574). In another embodiment, polymeric materials can be used (seeMedical Applications of Controlled Release, Langer and Wise (eds.), CRCPres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989,Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Othercontrolled-release systems discussed in the review by Langer, 1990,Science 249:1527-1533) may be used.

Dosages

The amount of the compound of the invention, or pharmaceuticalcomposition thereof which will be effective in the treatment, preventionor management of a pain, fever, inflammation, platelet-aggregation,colon cancer, pancreatic cancer, thrombotic or hemorrhagic disease ordisorder or other disease or disorder that can be treated, prevented ormanaged by administration of a compound of the invention can bedetermined by standard research techniques. For example, the dosage thatwill be effective in the treatment, prevention or management of ahemorrhagic or thrombotic disease or disorder can be determined byadministering the compound of the invention, or pharmaceuticalcomposition thereof to an animal model such as, e.g., the animal modelsdisclosed herein or known to those skilled in the art. In addition, invitro assays may optionally be employed to help identify optimal dosageranges. Selection of the preferred effective dose can be determined viaclinical trials.

The precise dose to be employed will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Animal models include but are not limited to those disclosed in Reyers,I., et al., 1985, Thromb. Haemost. 54(3):619-621; Louie, S, andGurewich, V., 1983, Thromb. Res. 30(4):323-35; Hahn D., et al., 1982,Prostaglandins 23(1):1-16; Koster J., et al., 1981, Artery 9(6):405-413;Ehard, H. et al., 1976, Z. Rheumatol. 35(9-10):324-336 and Russell, R.,et al., 1981, Scand J. Gastroenterol Suppl. 67:215-217.

Examples of thrombotic animal models include, but are not limited tothose in rats (Ravanat, C., et. al., Thromb. Haemost. 83:327-33 (2000)),rabbits (Zhao, et al., Brain Res. 902:30-9 (2001)), dogs (Bernat A.,1999, et. al., J. Cardiovasc. Pharmacol. 33:897-904), and baboons(Kruithof, E. K., 1997, et. al., Thromb. Haemost. 77:905-10).

In a preferred embodiment, the effective amount of salicylic acidadministered to a patient in need thereof is about 100 to 200 μg/ml. Inanother preferred embodiment, the effective amount of acetylsalicylicacid administered to a patient in need thereof is about 600 mg/ml. Inanother embodiment, the effective amount of acetylsalicylic acidadministered to a patient in need thereof is about 0.75 to 1.5grams/day.

In particularly preferred embodiments, the effective amount of aspirinadministered to a patient to treat or manage an ischemic event or atransient ischemic attack is 50-325 mg/day. In another preferredembodiment, the effective amount of aspirin administered to a patient totreat or manage a suspected myocardial infarction is 160-162.5 mg/dayfor 60 days.

In another preferred embodiment, the effective amount of aspirin,administered to a patient to treat, manage or prevent a recurrence ofmyocardial infarction, unstable angina pectoris, chronic stable anginapectoris, or coronary artery bypass grafting surgery is 75-325 mg/dayindefinitely. In another preferred embodiment, the effective amount ofaspirin administered to a patient to improve the therapeutic outcomeafter a carotid endarterectomy is 80-650 mg, twice daily. In anotherpreferred embodiment, the effective amount of aspirin administered to apatient to treat, manage or prevent rheumatoid arthritis, arthritis,osteoarthritis, or pleurisy is 3 g/day, initially. In another preferredembodiment, the effective amount of aspirin administered to a patient totreat, manage or prevent juvenile rheumatoid arthritis is 90-130mg/kg/day. In another preferred embodiment, the target level in theblood plasma of a patient.

In preferred embodiment, the effective amount of stabilized TAFIa is anamount which results in TAFI plasma levels in the patient's blood of0.01-20 μg/ml, preferably between 3-7 μg/ml, most preferably between0.01-10 μg/ml.

In a preferred embodiment, the effective amount of other compounds ofthe invention are 50 mg/day to 5,000 mg/day.

The following examples are offered by way of illustration, notlimitation.

EXAMPLES Example 1 TAFIa Inhibition by Acetylsalicylic Acid, SalicylicAcid and Salicyluric Acid

Materials. TAFI was purchased from Hematological Associates (EssexJunction, Vt.) and TAFIa (3874TAFIa) was from American Diagnostica Inc.(Greenwich, Conn.). Carboxypeptidase activity of TAFIa was determinedusing Actifluor™ TAFI (#874FX) a fluorescence-based assay from AmericanDiagnostica (Greenwich, Conn.). ASA, SA and SU were obtained from SigmaChemical Co. (St. Louis, Mo.).

Results. The present inventors have made the surprising discovery thatASA, SA and SU may shift the hemostatic balance toward a profibrinolyticstate by inhibition of TAFIa, the major regulator of fibrinolysis. TAFIawas incubated with various concentrations of ASA, SA and SU for onehour, at room temperature. Residual TAFIa activity was measured usingActiFLUOR TAFIa. ASA, as well as SA and SU, directly inhibited thecarboxypeptidase activity of TAFIa (FIG. 1). The rank order of potencywas SU>SA>ASA, indicating that the metabolites of ASA are more potentinhibitors than ASA itself. Interestingly, other NSAIDS, such asibuprofen and acetominophen, had little inhibitory effect on TAFIaactivity (see FIG. 4). The inhibition of TAFIa by the salicylates wasfound to be non-competitive (see FIG. 4), suggesting that they interactwith TAFIa outside the catalytic site of the enzyme. Catella-Lawson andcoworkers recently reported that ibuprofen, taken prior to aspirin,blocks the irreversible inhibition of COX-1 by aspirin and abrogates theanti-platelet activity of aspirin. Catella-Lawson, F. et al., 2001, N.Engl. J. Med. 345(25):1809-17. Since ibuprofen has little effect onTAFI, it was reasonable to conclude that ibuprofen taken prior toaspirin would not block the profibrinolytic activity of aspirin causedby TAFI inhibition.

Significant inhibition of TAFI activity occurs-around 100 to 200 μg/mlof SA and SU and 600 μg/ml for ASA (FIG. 1). Pharmacokinetic studiesshow that the concentrations of ASA and its metabolites, SA and SU,required to inhibit TAFI are readily achieved in plasma during high doseaspirin therapy (0.75-1.5 grams per day). Thus, bleeding complicationsof aspirin associated with physiologically high plasma concentrations ofASA, SA and SU correlate with inhibition of TAFI activity.

The results in FIG. 4 indicate that physiological concentrations of SAand SU also inhibit activation of TAFI to TAFIa bythrombin/thrombomodulin. Not only, then, do SA and SU inhibit enzymeactivity, they also retard the generation of TAFIa. Without being boundby any theory, this provides a dual mechanism by which fibrinolysis isenhanced during high dose aspirin therapy. The proposed pathways bywhich aspirin and its major metabolites SA and SU enhance fibrinolysisthrough inhibition of TAFI is presented in FIG. 5. Undas recently showedthat thrombin generation at the wound site is also inhibited by aspirin.Undas et al., 2001, Blood 98:2423-31. This suggests that aspirin therapymay also affect other components of the coagulation pathway.

Example 2 Synthesis of Chemical Compounds

This example provides pathways for synthesizing some of the compounds tobe tested in the assay described in Example 3.

Synthesis of 2-(2-hydroxybenzoylamino)ethyl N-(3-aminopropyl)carbamatea278R)

N-(2-Hydroxyethyl)-2-hydroxybenzamide (5.43 g, 30 mM) was dissolved indry pyridine (40 ml). The reaction mixture was vigorously stirred at 0°C. while p-nitrophenyl chloroformate (9.0 g, 45 mM) was added inportions over 30 min. The reaction mixture was stirred at r.t. for 20hrs and the solvent was removed under vacuum. The residue was dissolvedin CHCl₃ and chromatographed on a silica gel column withCHCl₃:EtOH=99:1. The fraction preceding the yellow zone was collectedand evaporated to dryness to give O-(2-hydroxybenzoylamino)ethylO′-(4-nitrophenyl)carbonate as a colorless solid (8.0 g).O-(2-hydroxybenzoylamino)ethyl O′-(4-nitrophenyl)carbonate (345 mg) wasdissolved in dry DMF (2 ml), 3-azidopropylamine (500 mg, 5 mM) was addedand the reaction mixture was stirred at r.t. for 48 hrs. The volatileswere removed under vacuum (0.2 mm Hg) and the residue waschromatographed on silica gel column with hexane:ethyl acetate=3:1 to2:1. The only UV fluorescent zone afforded after removal of the solventunder vacuum 2-(2-hydroxybenzoylamino)ethyl N-(3-azidopropyl)carbamate(220 mg) as a colorless solid.

2-(2-Hydroxybenzoylamino)ethyl N-(3-azidopropyl)carbaniate (128 mg) wasdissolved in methanol (15 ml), 10% Pd/C (140 mg) was added and thereaction mixture was vigorously stirred while hydrogen was bubbled atr.t. for 3 hrs. The catalyst was filtered off and the methanol removedunder vacuum to provide 2-(2-hydroxybenzoylamino)ethylN-(3-aminopropyl)carbamate (88 mg) as a colorless oil.

Synthesis of 2-(2-hydroxybenzoylamino)ethylN-2-[2,3-bis(benzyloxy)benzoylamino]ethyl carbamate (LT32)

2-[2,5-Bis(benzyloxy)benzoylamino]-ethylamine (564 mg, 1.5 mM) andO′-[2-(2-hydroxybenzoylamino)-3-phenyl-propyl]-O′-(4-nitrophenyl)carbonate(522 mg, 1.5 mM) were dissolved in dry DMF (4 ml) and the mixture wasstirred at r.t. for 24 hrs. The solvent was removed under vacuum and theresidue was chromatographed on a silica gel column with hexane:ethylacetate=50:50, 33:66, 20:80. The fractions containing the product werecombined, the solvent removed under vacuum and the residue wasrecrystallized from hexane:ethyl acetate and once again from ethylacetate to provide 2-(2-hydroxybenzoylamino)ethylN-2-[2,3-bis(benzyloxy)benzoylamino]ethyl carbamate (500 mg) as acolorless solid.

Synthesis of N-[(S)-1-hydroxy-3-phenyl-2-propyl]-2,5-dihdroxybenzamide(JV59)

In a flask equipped with a reflux condenser and a magnetic stirring barare dissolved the methyl 5-benzyloxy-2-hydroxybenzoate and oneequivalent of 2-(S)-amino-3-phenyl-1-propanol in dry dimethyl formamide.The flask is immersed in an oil bath at 75° C. and left to stir for 72hours. The solvent is evaporated and the residue is isolated-through theuse of a silica gel column using a mixture of ethyl acetate and hexaneas eluent. In this fashion is obtained in 50% yield,N-{(S)-1-hydroxy-3-phenyl-2-propyl}-5-benzyloxy-2-hydroxybenzamide.

In a flask equipped with a magnetic stirring bar and a rubber septum isdissolved theN-{(S)-1-hydroxy-3-phenyl-2-propyl}-5-benzyloxy-2-hydroxybenzamide inmethanol. Nitrogen is bubbled through the solution and 10% Pd oncharcoal is added. After flushing with nitrogen, the suspension isplaced under hydrogen atmosphere and stirred for 2 hours. The suspensionis flushed with nitrogen and filtered and rinsed over Celite. Thefiltrate is concentrated under vacuum. In this fashion is obtainedN-{(S)-1-hydroxy-3-phenyl-2-propyl}-2,5-dihydroxybenzamide (85%).

Example 3 Measurement of Compounds for TAFI-Iinhibition

The activity of the chemical compounds was assessed in the ActiScreen®TAFI HTS assay, the principle of which is shown below.

The assay is run by mixing TAFIa and test compound in microtiter platewells. “TAFI developer” is then added to start the reaction which is runat room temperature. The TAFI developer is formulated withhippuryl-L-lysine, lysine oxidase, horseradish peroxidase andortho-phenylene-diamine. Hippuryl-L-lysine is converted by TAFIa tohippuric acid and L-lysine. Lysine-Oxidase (LOX) converts the lysine to6-amino-2-oxo-hexanoic acid and hydrogen peroxide. The hydrogen peroxideis used along with ortho-phenylene-diamine (POD) as substrate forhorseradish peroxidase. The reaction produces a yellow color that wasmonitored for Vmax in a spectrophotometer (SpectroMax 190, MolecularDevices, Sunnyvale, Calif.) in the kinetic mode at 450 nm.

The assay can also be run as an endpoint reaction in which the enzymaticreaction is stopped at a selected point in time with 0.5 M sulphuricacid. The resulting color is read at 490 nm. A TAFI inhibitor will blockthe first reaction in the cascade in which TAFIa convertshippuryl-L-lysine to hippuric acid and lysine, which finally results ina decrease in Vmax or OD 490 nm relative to control reaction withoutaddition of test compound. The assay has been designed to be run infinal concentrations of DMSO of up to 20%. A known inhibitor of TAFIacarboxypeptidase activity, potato tuber carboxypeptidase inhibitor(PTCI), is used as a positive control.

The TAFIa used in the ActiScreen HTS assay is a specially formulatedpreparation from native human TAFI which is highly more stable thanTAFIa prepared by conventional methods. The enhanced ability ofstabilized TAFIa at room temperature compared to conventional TAFIapreparation is shown in FIG. 7.

Example 4 Screening Chemical Compound Libraries

Positive hits were selected based upon the ability of compounds toinhibit carboxypeptidase activity of TAFIa, as measured in theActiScreen assay. The major criterion for selection of “hits” was basedon their potency, as determined by the concentration of compound thatinhibits the activity of TAFIa in the Actiscreen TAFI assay by 50%(“IC50”).

Positive hits identified with the ActiScreen TAFI assay weredereplicated to ensure that the inhibition of color development is dueto blocking of the first reaction of the linked enzyme system catalyzedby TAFIa and not to inhibition of LOX or HRP. Dereplication wasperformed by testing whether the lead test compounds inhibit thereaction of LOX/HRP with d/l -lysine as the substrate. All compoundswere tested at concentrations 5-10 times greater than their IC50 valuesdetermined for TAFI.

Using this methodology, the inventors have screened hundreds compoundsand identified structural motifs that are associated with inhibition ofTAFIa carboxypeptidase. One class of TAFI inhibitors is represented bythe compound L278R, which inhibits TAFIa via two mechanisms. When theassay was performed as described in Example 3, L278R appeared to be acompetitive type inhibitor of TAFI with an IC50 value of 1.54 mM.However, when TAFIa was preincubated with L278R for 1 hour at 4° C.prior to testing for carboxypeptidase activity using ActiScreen TAFIassay, the IC50 shifted to a significantly lower value 0.89 mM (FIG. 8).This shows that L278R also can act as a non-competitive-type inhibitorof TAFI. The structure of L278R is shown below.

Another class of TAFI inhibitors is represented by the compound LT32.The IC50 determined for LT32 is 225 μM. LT32 is a competitive inhibitorof TAFIa (FIG. 9). The structure of LT32 is shown below.

The compound JV59 has structural features in common with the class ofcompounds to which LT32 belongs. JV59 scored as a potent hit in thescreening assay, with an IC50 of 44 μM. The structure of JV59 is shownbelow.

Compounds L278R and LT32 were tested in the dereplication assay,described above. Neither of the compounds had a significant effect onthe reaction rate (Vmax) of LOX/HRP with lysine. The inventors concludethat these compounds inhibit TAFIa and not LOX/HRP.

L278R was also tested for specificity against Carboxypeptidase N(CPN).CPN is the constituitively active carboxypeptidase B-like enzyme inplasma. In this study, pooled normal human plasma (PNP) was used as thesource of CPN. The specificity studies were performed by adding PNP(1:10), ActiScreen developer and serially diluted test compound intomicrotiter wells. Inhibition of color development was an indication ofinhibitory activity towards CPN. The results indicated that L278R didnot inhibit CPN, demonstrating that it is specific for TAFIa.

Example 5 Screening Compounds for Analgesia

A test compound, such as a derivatized analgesic, is selected. Thecompound is incubated with TAFIa for 30 minutes and TAFIa inhibition ismeasured using a fluorometric in vitro assay as described herein. If thecompound shows little or no TAFIa inhibition, then the test compound isadministered to a rat prior to conducting a hotplate assay as describedherein. If the latency to the lick is increased by the test compound,then a non-inhibitor of TAFIa with potential analgesic activity isidentified. The identified compound may then be tested for otheractivities, e.g., antipyretic activity.

Many modifications and variations of the present invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled

1. A method of identifying a compound which possesses (i) at least oneaspirin activity selected from the group consisting of analgesia,anti-inflammation, fever reduction, COX-1 inhibition, COX-2 inhibition,anti-platelet aggregation and pancreatic cancer prophylaxis, and (ii) noTAFIa-inhibition, comprising the steps of: a) exposing TAFIa to the testcompound; b) determining whether the test compound inhibits TAFIaactivity and/or inhibits TAFI activation; and c) determining whether atleast one aspirin activity is retained by the test compound, therebyidentifying a compound that possesses at least one aspirin activity butnot TAFIa-inhibitory activity.
 2. The method of claim 1, wherein theaspirin activity is COX-2 inhibition.
 3. The method of claim 1, whereinthe aspirin activity is COX-1 inhibition.
 4. The method of claim 1,wherein the aspirin activity is analgesia.
 5. The method of claim 1,wherein the aspirin activity is fever reduction.
 6. The method of claim1, wherein the aspirin activity is anti-inflammation.
 7. The method ofclaim 1, wherein the aspirin activity is anti-platelet aggregation. 8.The method of claim 1, wherein the aspirin activity is colon cancerprophylaxis.
 9. The method of claim 1, wherein the aspirin activity ispancreatic cancer prophylaxis.
 10. A method of treating pain, fever,inflammation, platelet-aggregation, colon cancer, pancreatic cancer,fibrinolytic, or thrombotic disease or disorder in a patient in needthereof, comprising administering a compound identified by the method ofclaim 1 to the patient.
 11. A method of evaluating inhibition of TAFIaactivity by a test compound, in comparison with aspirin, salicylic acid,salicyluric acid or gentisic acid, comprising the steps of: a) exposingTAFIa to the test compound; b) measuring inhibition of TAFIa activity bythe test compound; and c) comparing the inhibition of TAFIa activity bythe test compound to inhibition of TAFIa activity by aspirin, SA, SU orgentisic acid, thereby determining whether the test compound inhibitsTAFIa activity more or less than aspirin, salicylic acid, salicyluricacid or gentisic acid.
 12. A compound identified by the method of claim11.
 13. The method of claim 11, wherein an inhibitor of TAFIa isidentified if the test compound inhibits TAFIa activity as compared toaspirin, salicylic acid, salicyluric acid or gentisic acid.
 14. Themethod of claim 11, wherein step (a) further comprises preincubatingTAFIa and the test compound for at least about 20 minutes.
 15. Themethod of claim 11, wherein step (b) is carried out using a clot lysisassay.
 16. The method of claim 11, wherein step (b) is carried out usinga fibrinogen assay.
 17. The method of claim 11, wherein step (b) iscarried out using a plasminogen capture assay.
 18. The method of claim11, wherein step (b) comprises using a fluorescent based assay.
 19. Amethod of inhibiting TAFIa carboxypeptidase activity in a patient inneed thereof, comprising administering to the patient a pharmaceuticalcomposition comprising a TAFIa inhibiting compound selected from thegroup consisting of acetylsalicylic acid, salicylic acid, salicyluricacid, gentisic acid, glycyl amides and α-hydroxybenzoate derivatives, inan amount effective to inhibit TAFIa activity.
 20. The method of claim19, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 21. A method of treating ormanaging a thrombotic disorder in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of aninhibitor of TAFIa, wherein said inhibitor is selected from the groupconsisting of salicylic acid, salicyluric acid, gentisic acid, glycylamides, acetylsalicylic acid and α-hydroxybenzoate derivatives, in anamount effective to inhibit TAFIa activity.
 22. The method of claim 21,wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 23. The method of claim 21, whereinthe thrombotic disease or disorder is selected from the group consistingof angina pectoris, chronic stable angina pectoris, heart attack,stroke, thromboembolic disease, myocaridal infarction (MI), acutemyocardial infarction (AMI), recurrent myocardial infarction, ischemicattack, transient ischemic attack (TIA), deep vein thrombosis, acuteischemic stroke, massive pulmonary embolism, disseminated intravascularcoagulation (DIC), anti-phospholipid syndrome, familial thrombophilia,sepsis, arthritis, fulminant hepatitis, thrombosis, hemophilia and VonWillebrand disease.
 24. A method of preventing a thrombotic disorder ina patient, comprising administering to the patient a prophylacticallyeffective amount of an inhibitor of TAFIa, wherein said inhibitor isselected from the group consisting of salicylic acid, salicyluric acid,gentisic acid, glycyl amides, acetylsalicylic acid and α-hydroxybenzoatederivatives.
 25. The method of claim 24, wherein said inhibitor of TAFIais formulated with a pharmaceutically acceptable carrier.
 26. The methodof claim 24, wherein the thrombotic disorder is angina pectoris, chronicstable angina pectoris, heart attack, stroke, thromboembolic disease,myocaridal infarction (MI), acute myocardial infarction (AMI), recurrentmyocardial infarction, ischemic attack, transient ischemic attack (TIA),deep vein thrombosis, acute ischemic stroke, massive pulmonary embolism,disseminated intravascular coagulation (DIC), anti-phospholipidsyndrome, familial thrombophilia, sepsis, arthritis, fulminanthepatitis, or thrombosis.
 27. A method of reducing bleeding side-effectsof aspirin in a patient, said method comprising administering stabilizedTAFIa to a patient receiving aspirin therapy.
 28. The method of claim27, wherein aspirin is administered prior to, subsequent to, orconcurrently with said administration of stabilized TAFIa.
 29. Themethod of one of claims 19, 21, 24 or 27 further comprising surgery onthe patient.
 30. The method of claim 29, wherein the surgery is selectedfrom the group consisting of organ transplant surgery, cardiopulmonarybypass surgery, coronary angioplasty, open heart surgery, heart valvesurgery and removal of atherosclerotic plaques.
 31. The method of one ofclaims 19, 21, 24, 27-30 further comprising administering ananti-coagulant to the patient.
 32. The method of claim 31, wherein theanti-coagulant is selected from the group consisting of tissueplasminogen activator, heparin, low molecular weight heparin, aspirin,actothrombin, hirudin, plasminogen, streptokinase, staphylokinase,urokinase and hirulong. 33-37. (canceled)
 38. A method of treating ormanaging a thrombotic disorder in a patient in need thereof, comprisingadministering to the patient a therapeutically effective amount of apharmaceutical composition comprising a compound having the chemicalstructure selected from the group consisting of:

in an amount effective to inhibit TAFIa activity.
 39. The method ofclaim 38, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 40. The method of claim 38, whereinthe thrombotic disease or disorder is selected from the group consistingof angina pectoris, chronic stable angina pectoris, heart attack,stroke, thromboembolic disease, myocaridal infarction (MI), acutemyocardial infarction (AMI), recurrent myocardial infarction, ischemicattack, transient ischemic attack (TIA), deep vein thrombosis, acuteischemic stroke, massive pulmonary embolism, disseminated intravascularcoagulation (DIC), anti-phospholipid syndrome, familial thrombophilia,sepsis, arthritis, fulminant hepatitis, thrombosis, hemophilia and VonWillebrand disease.
 41. A method of preventing a thrombotic disorder ina patient, comprising administering to the patient a pharmaceuticalcomposition comprising a compound having the chemical structure selectedfrom the group consisting of:

in an amount effective to prevent the thrombotic disorder.
 42. Themethod of claim 41, wherein pharmaceutical composition further comprisesa pharmaceutically acceptable carrier.
 43. The method of claim 41,wherein the thrombotic disorder is selected from the group consisting ofangina pectoris, chronic stable angina pectoris, heart attack, stroke,thromboembolic disease, myocaridal infarction (MI), acute myocardialinfarction (AMI), recurrent myocardial infarction, ischemic attack,transient ischemic attack (TIA), deep vein thrombosis, acute ischemicstroke, massive pulmonary embolism, disseminated intravascularcoagulation (DIC), anti-phospholipid syndrome, familial thrombophilia,sepsis, arthritis, fulminant hepatitis, or thrombosis.
 44. The method ofone of claims 38 or 41, further comprising surgery on the patient. 45.The method of claim 44, wherein the surgery is selected from the groupconsisting of organ transplant surgery, cardiopulmonary bypass surgery,coronary angioplasty, open heart surgery, heart valve surgery andremoval of atherosclerotic plaques.
 46. The method of one of claims 38or 41, further comprising administering an anti-coagulant to thepatient.
 47. The method of claim 46, wherein the anti-coagulant isselected from the group consisting of tissue plasminogen activator,heparin, low molecular weight heparin, aspirin, actothrombin, hirudin,plasminogen, streptokinase, staphylokinase, urokinase and hirulong.